E N Z Y IKHIBITORS. ~~~E VI11
July 1965
9-position a n-propyl group, a 3-hydroxypropyl group, a 2-hydroxypropyl group, and a 2,3-dihydroxypropyl group. Chemistry.-The synthesis of 6-amino- and 6methylamino-9-n-propylpurine (I and 11) mas accomplished by the method of Teinple, et uL16 and the preparation of 6-amino- and 6-methylaniino-9-(3hydroxypropy1)purine (111and IV) mas effected by the method described previously. The syntheses of the 6-substituted 9-(2-hydroxypropyl)purines and the 6437
R
R
I, R = NH, 11, R = NHMe
111, R =KH, IV, R = NHbIe
substituted 9-(2,3-dihydroxypropyl)purines were accomplished by the routes outlined in Charts I and 11, respectively. Thus, when 5-aniin0-4~6-dichloropyrimidine (V) was allowed to react with 1-amino-2-propatio1 (VI), a good yield of the corresponding substitut ed pyriniidine T'II was obtained. Cyclization of VI1 by iiieaiis of ethyl orthoforniate aiid hydrochloric acid gave the desired 6-chloropurine derivative VIII. The 6-subst ituted 9- (2-hydroxypropy1)purines (IXXIV) were prepared by allowing VI11 to react with the appropriate nucleophilic reagent.
503
Thus, when XVII was converted into XVIII by nieans of hydrogen chloride, and XVIII allowed to react with iiiethanolic aiiiino~iia,B-aniino-9-(2,3-dihydroxypropyl)purine (XIX) mas produced. This 6-amino derivative (XIX) was identical i n all respects with the product formed by allowing XVII to react with niethariolic ammonia to give XXIII which on treatment with HCI gave XIX. This sequence of reactions t o produce XIX is consisterit with the assignment of the orthoester structure to XVII. The formation of an orthoester froin a vicinal diol on a substituent at the 9position of a purine nucleus suggests that this reaction may find utilization in the formation of a new type of blocking group for riboinwleosides arid perhaps for ribonucleotides. Recently, it has been reported that certain ribofuranosylpurines are, in fact, converted into the corresponding orthoesters by reaction with ethyl orthoformat e.8, CHARTI1
V
CH2 I CHOH
I
CHzOH XVI
NHz
J
Cl
CHARTI
c1
CHzNHz
-k CHOH
I
-
+
N'
CH,
I
ANHCH~CHOHCH,
VI
VI1
XXIII
R
\IlI, IX, X, XI,
I
CHz
K = c1 XII, K = SH R = NH2 XIII, K = OH R = NHXIe XIT', R = OMe R = NMe,
XVII
C Ha
CH2
I
I
CHOH
similar s e q u e n c ~of~ reacstioiia was eiuployrd for t preparation of the 6-substitutcd 9-(2,3-dihydroxypropy1)puriiies (Chart 11). Condensation of V with l-ainino-2,3-dihydroxypropane(XV) gave 5-amino-4chloro-6-(2,3-dihydroxypropylaiiii1io)pyrii~iidine (XVI) in an excellent yield. Attempted cyclization of XT'I to produce (i-chloro-9-(2,3-dihydroxypropyl)purine (XVIII) by nieans of ethyl orthoforniate arid HCl or by niearis of diethoxyiiiethyl acetate gave a conipound which 011 the basis of its ultraviolet spectruni was a 6-chloropurine derivative but which did not exhibit absorption in its infrared spectrum for hydroxyl groups. However, on the basis of its subsequent conversion into XVIII and XXIII, we have assigned structure XVII to the initial ryclization product. I i ( 5
(6) C. Temple, Jr. C. L. Kussner, and J. .L l l o n t g o m e r y , J . M e d . Pharm. Chem., 6, 866 (1962). (7) hl. Ikehara, E. Ohtsuka, S. K i t a g a u a , K. Yagi, and Y. Tonomura, J . A m . Chem. Sac., 85, 2679 (1961).
I
CHzOH XIX, R = NHz XX, R = N H M e XXI, R=NMe2 XXII, R = SH
CHOH
I
CHzOH XVIII
Experimental'" 5-Amino-4-chloro-6-(P-hydroxypropylamino)pyrimidine (VII). -A solution of 4.43g. (27.0 mmoles) of V, 2.07 g. (37.5 mmoles) of 1-amino-2-propanol, and 3.04 g. (30.0 mmoles) of triethylamine i n 30 ml. of 1-butanol was heated under reflux for 21 hr. The volatile materials were removed i n vacuo and the oily product was dis(8) J. Zemlicka, Chem.lnd. (London), 581 (1964). (9) AT, J a r m a n a n d C . B. Reese, ibid., 1493 (1964). (10) The infrared spectra were determined on a Perkin-Elmer Alodel 137 spectrophotometer; the ultraviolet spectra and enzyme rates were determined on a Perkin-Elmer Model 4000A spectrophotometer. The melting points, unless noted otherwise, were determined on a Kofler Heizbank and are corrected.
July 1965
E N Z Y h l E ISHIBITORS.
matography R f values as the oil obtained by the cyclization procedure employing triethyl orthoformate. A small amount of this material was dissolved in CHCL and converted to 3-(6-chloro-9purinyl)-l,2-propanediol hydrochloride by bubbling HCl gas t,hrough the chilled solution and collecting the precipitate, m.p. 155-160'. XVIII was identical in every respect with an authentic sample prepared by a method previously described. 3 4 6-Amino-9-purinyl)-l,2-O-dihydroxypropyl Ethyl Orthoformate (XXIII).-A mixture of 219 mg. (1.00 mmole) of XVI, 6.0 ml. of triethyl orthoformate, and 1.30 mmoles of concent,rated HC1 was stirred overnight a t room temperature. The volatile materials were removed in vacuo a t 35' and 20 ml. of 19y0 methanolic NH, was added. The solution was placed in a steel bomb and heated overnight a t 55". Removal of the volatile materials gave a soft white solid. Chloroform (10 ml.) RTas added, and the hot mixture was filtered to remove NHJ.3. The filtrate was treated with hexane until cloudy and chilled overnight, which gave a fine white solid that was removed by filtrat'ion; yield 108 mg., m.p. 137-140' (oil bath). Recrystallization from ethyl acetatehexane gave 63.5 mg. (247,), m.p. 162-164'. A second run gave 377, of the pure material: ,A, in mp ( e x lod4),a t p H 1, 261 (1.49), a t p H 7 , 261 (1.54), a t pH 13, 261 (1.54); Y in em.-' (KBr), 3300 (NHz),1660 and 1590 (C=X and C=C). Anal. Calcd. for CllH15N503:C, 49.80; H, 5.69; N, 26.40. Found: C,49.65; H, 5.75; S, 26.46. 3-(6-Amino-9-purinyl)-1,2-O-dihydroxypropyl Ethyl Orthoformate (XXIII).-The 3-(6-chloro-9-purinyl)-1,2-O-dihydroxypropyl ethyl orthoformate (XVII, 1.07 g., 3.74 mnioles) obtained from the diethoxymethyl acetate cyclization was dissolved in 20 ml. of 207, methanolic XH3. The mixture was heated in a steel bomb a t 55' for 18 hr. Removal of the volatile materials in vacuo gave a semisolid material. Chloroform (20 ml.) was added and the NH4C1was removed by filtration. The addition of hexane to the filtrate caused the separation of 382 mg. (38.47,) of pure material, m.p. 160-163" (oil bath). The ultraviolet and infrared spectra were identical with those of an authentic sample prepared by a different procedure. Anal. Calcd. for CllHljNjO~:C, 49.80; H, 5.69; S, 26.40. Found: C, 49.95; H, 5.81; ?i, 26.73. 6-Amino-9-(2,3-dihydroxypropyI)purine Hydrochloride (XIX). -A solution of 250 mg. (0.942 mmole) of XXIII in 3 ml. of 107, HCI was left a t room temperature for 2.5 hr. Removal of the volatile constituent,s in vacuo a t 50' followed by the addition of methanol and ether gave t'he pure hydrochloride salt (XIX); in mp ( E x at, yield 212 mg. (91%); m.p. 216-219"; A,, p H 1, 260 (1.36), a t pH 7 , 261 (1.38), a t pH 13, 261 (1.42); Y in em.-' (KBr), 3350 (OH), 1670 (C=N+H), 1580 (C=C). Anal. Calcd. for CsH&1Nb02: C, 39.10; H, 4.92; K,28.50. Found: C, 38.84; H, 5.18; N, 28.10. 6-Amino-9-(2,3-dihydroxypropyl)purineHydrochloride (XIX). -A solution of 500 mg. (1.88 mmoles) of XVIII in 20 ml. of 207, methanolic NH3 was heated in a steel bomb a t 80" for 20 hr. Removal of the volatile materials in ~ a c u ogave 588 mg. of a soft yellow solid. The crude solid was dried to a brittle material in vacuo, but quickly softened on exposure t o air. A hydrochloride salt was prepared by dissolving the crude material in anhydrous methanol and passing dry HC1 through the chilled solution. The white precipitate was collected by filtration and two recrystallizations from methanol-ether gave the analytical material XIX, yield 260 mg. (56.1%), m.p. 215-218'. The melting point and infrared and ultraviolet spectra were identical with those of an authentic sample prepared by a different procedure. B-Methylamino-9-( 2,3-dihydroxypropyl)purine Dihydrochloride (XX).-A solution of 500 mg. (1.88 mmoles) of XVIII in 20 ml. of 40% methylamine in water was heated under reflux for 3 hr. The volatile materials were removed in vacuo and gave a yellow oil. The oily mixture was dissolved in 10 ml. of absolute ethanol and methylamine hydrochloride was removed by adding ether or chloroform and removing the salt by filtration. To the filtrate was added CHCla (10 ml.) and HC1 was bubbled through the chilled solution until a precipitate formed. Ether (20 ml.) was then added, and the white solid was collected by filtration; yield 492 mg., m.p. 183-186" (oil bath). The material was purified by dissolving in methanol (10 ml.) and bubbling HCl through the chilled solution until a precipitate formed. Ether (15 ml.) was added, and the pure product X X was collected by filtration: yield 394 mg. (70.8%); m.p. 190-196" (oil bath); A,, in mp ( E x a t pH 1,263 (1.74), a t pH 7,266 (1.65), a t pH 13,266 (1.65); pin cm.-l(KBr), 3350 (OH), 3000-2750 (XH3+), 1675 (C=N+H), 15% (C=C).
VI11
505
Anal. Calcd. for C9HljCI2N5O2:C, 36.49; H, 5.10; N, 23.65. Found: c, 36.25; H, 5.26; N, 23.75. 6-Dimethylamino-9-(2,3-dihydroxypropyl)purineDihydrochloride (XXI).-A mixture of 500 mg. (1.88 mmoles) of XVIII in 20 ml. of 25Y0 aqueous dimethylamine was heated under reflux for 3 hr. The volatile materials were removed in vacuo and methanol (5 ml.) was added to the residue. After the chilled solution was saturated with HC1 gas, CHCla (10 ml.) was added to the mixture. The white salt was removed by filtration, washed with CHC13, and gave 439 mg. (74.67,) of XXI, m.p. 164-167' (oil bath). A small sample was recrystallized from niethanol-ether saturated with HC1; m.p. 164-167";, , ,X in mp ( e X lod4),a t p H 1, 269 (1.54), at pH 7 , 275 (1.57), a t pH 13, 276 (1.57); Y in cm.-' (KBr), 3400 (OH), 2750 and 2350 (XHS ), 1665 (C=N+H), 1565 (C=C). Anal. Calcd. for CloH17C12Sj02: C, 38.71; H, 5.53; S , 22.58. Found: C, 38.72; H, 5.67; N, 22.31. 6-Mercapto-9-(2,3-dihydroxypropyl)purine (XXII).-A mixture of 500 mg. (1.88 mmoles) of XVIII and 152 mg. (2.00 mmoles) of thiourea in 20 ml. of n-propyl alcohol was heated under reflux for 2 hr. The reaction mixture was evaporated to about 5 ml. an DUCUO and CHC1, (10 nil.) was added. After being chilled, the mixture was filtered to remove the crude product and gave 319 mg. of a soft yellow solid. Recrystallization from 1-propanol gave 154 mg. (36.4%) of the pure material XXII: m.p. 285-288"; A, in mp ( e x a t pH 1, 323 (1.96), a t pH 7 , 321 (2.16), at pH 13, 310 (2.03); Y in em.-' (KBr), 3400 (OH), 2600 (SH), 1600 and 1540 (C=N and C=C). Anal. Calcd. for CRHION~O~S: C, 42.46; H, 4.45; S, 14.17. Found: C, 42.19; H, 4.68; S, 13.91. Reagents and Assay Procedure.-Adenosine and adenosine deaminase mere purchased from the Sigma Chemical Co. The general method of assay has been described previously. l 3 All enzymatic reactions were run in phosphate buffer (0.05 M ) a t pH 7.6 and a t 25". Solutions of the enzyme, substrate, and inhibitors were all prepared in 0.05 M phosphate buffer a t pH 7.6. During each experiment, the cells contained a total volume of 3.1 ml. which was 0.066 m31 with respect to adenosine. Sufficient amounts of enzyme were used so that the initial rate of reaction gave a change of approximately 0.6 optical density units/min. Compounds which did not show significant inhibition at concentrations 2-3 times that of substrate nere classified as noninhibitory. Inhibitors were compared by means of the inhibition index [ I / S ] O {.e., the ratio of the m d l concentration of inhibitor to themM concentration of substrate for 50% inhibition. In order to determine the concentration of an inhibitor required for 50% inhibition, a plot of VO/Vus. I was made where V O= initial velocity of the uninhibited enzymatic reaction, V = initial velocity of the inhibited enzymatic reaction a t various inhibitor concentrations, and I = the various concentrations of inhibitor.14
Results Erizyiiiatic cvaluat ion of these purine derivatives revealed that for any given 9-substituent, those conipounds with an amino group at the 6-position n-ere more inhibitory than those coiiipounds with a 6methylamino group which in turn were inore inhibitory than those compounds with a 6-diniethylami1io group. Those compounds with a 6-chloro, a 6-niercapto, a 6hydroxy, or a 6-methoxy group were essentially 11011inhibitory. These results are in agreement with the data previously found concerning the ability of various groups a t the 6-position of the purine nucleus to bind to adenosine deaminase.'; However, when a comprtrison is made for a given substituent at the 6-position of the purine nucleus, then it is found that the inhibition decreases in the following order for the 9-substituent : 2-hydroxypropyl > 3-hydroxypropyl > 2,3-dihydroxypropyl > propyl. The constants for the various coinpounds are given in Table I. (13) iX.0. Kaplan in "RIethods in Enzymology," Vol. 11, S. P. Colouick and N. 0. Kaplan, Ed., ilcademic Press Inc.. New Pork, N. Y . , 1955, p. 473. (14). B. R. Baker and H. S.Sachder, J . Pharm. Scz., 6 2 , 933 (1963). (15) H. J. Schaeffer and R. Vince, J . M e d . Chem.. 8, 33 (1965).
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x
